The long-term objective of the research program is to develop the use of substitution-inert metal ions as effective probes of structure-function relationships in biological molecules. Direct investigation of the interactions of these metal ions with biological molecules and their analogues are being studies. Attention is being focused on imidazole coordination as there is a preponderance of histidine side chains involved in native metal ion binding sites in enzymes. Peptide chelate analogues of these sites are being assembled and will be subjected to proteolysis to determine the stability of cooresponding protein chelates during similar excision experiments. These studies are being undertaken with the expectation that a better method will be developed to determine the nature of metal ion binding sites in proteins, a problem that has consumed a considerable amount of effort and expense in the past. The site-specific modification of arsanilazotyrosine-248 carboxypeptidase A with substitution-inert metal ions is being investigated in order to determine whether this residue is essential to catalysis. This chelate will also be excised to establish its identity by comparison with previously synthesized analogues. This approach to protein modification is being investigated for its potential as a general method for incorporating specific metal ion probes into biological molecules.